JPH11315096A - Antitumoral protein derived from pleurotus cornucopiae and its gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a protein having activities selectively inhibiting the propagation of a tumor cell, and a gene therefor. SOLUTION: This protein derived from a fruit body of Pleurotus cornucopiae is salted out by 60% ammonium sulfate, has 150-200 kDa estimated molecular weight measured by a gel-filtration method, about 200 kDa estimated molecular weight measured by HPLC gel filtration, and the maximum absorption at about 277 nm wave length in an ultraviolet absorption spectrum. The protein is precipitated and the antitumoral activities are decreased at >60 deg.C. The protein has stable antitumoral activities at pH 3-12.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an antitumor protein derived from the fruit body of Pleurotus cornucopiae , a recombinant protein thereof, a gene thereof, and an antitumor agent containing the protein as an active ingredient.

[0002] has been done many from research on the anti-tumor substance of the background art edible mushrooms earlier, the fruiting body of the mushroom, mycelium, it is known that the presence of anti-tumor substances in bagasse and the like. For example, Japanese Patent Publication No. 54-27917, Japanese Patent Publication No. 60-1868
Japanese Patent Publication No. 53-136511 and Japanese Patent Publication No. 51-13631, Japanese Patent Publication No. Sho 53-203651, and Japanese Patent Publication No. Sho 62-209091. No. 167 includes Japanese Patent Publication Nos. 49-484 and 54-1 from Shimeji.
No. 7186 and JP-B-56-46818 describe antitumor polysaccharides and glycoproteins derived from Shiitake mushroom, and Japanese Patent Publication No. 2-78630 describe Kawariaritake mushroom. In addition, it has been reported that mushrooms were orally administered, and antitumor activity was observed.

[0003] However, as far as the present inventors are aware, there is no report of a protein derived from Hemophilus mushroom which directly acts on cancer cells and its gene.

[0004]

SUMMARY OF THE INVENTION The present inventors have now found that among commercially available edible mushrooms, Pleur otus cornucopia
It was found that a protein having a particularly high antitumor activity was present in the buffer extract of e ), and the protein was successfully isolated and purified. The present inventors have also succeeded in isolating a gene encoding an antitumor protein from a chromosome library derived from the fruit body of Pleurotus cornucopiae and expressing the gene in Escherichia coli. The present invention is based on this finding.

Accordingly, an object of the present invention is to provide an antitumor protein and a recombinant protein thereof, a gene encoding the protein, a pharmaceutical composition containing the protein, and a method for producing the protein.

[0006] The protein according to the present invention is an antitumor protein derived from the fruit body of Pleurotus cornucopiae, which has the following properties (1) to (5): (1) 60% ammonium sulfate (2) Estimated molecular weight by gel filtration method is 150 to 200 kDa, estimated molecular weight by HPLC is about 200 kDa. (3) In the ultraviolet absorption spectrum, maximum absorption is observed at a wavelength around 277 nm. (4) 60 (5) has a stable antitumor activity in the range of pH 3 to 12, which precipitates at a temperature exceeding ℃ and reduces the antitumor activity.

The recombinant protein according to the present invention has (a) the amino acid sequence of SEQ ID NO: 1 or (b) one or more additions, insertions, substitutions, and / or deletions, and has antitumor activity. Having the amino acid sequence of SEQ ID NO: 1 having

[0008] The protein according to the present invention is useful as an antitumor agent.

[0009]

DETAILED DESCRIPTION OF THE INVENTION Antitumor protein The protein according to the present invention has tumor cell growth inhibitory activity. The protein according to the present invention can be used not only for cancerous BALB mouse normal cells but also for human cervical cancer, He
La cells, SV40 transformed cells derived from human normal lung tissue WI-38 VA13subline 2RA, mouse leukemia cells P388D1
And also acts on human acute lymphocytic leukemia cells MOLT-4.

[0010] The antitumor protein according to the present invention is also transformed by SV40, which causes cancer in mice by the same mechanism of action as human papilloma virus (HPV), which is the virus responsible for squamous cell carcinoma represented by human uterine cancer. It shows cytotoxicity only to (cancerized) mouse embryonic fibroblasts, and does not show cytotoxicity to untransformed normal cells. That is, the protein according to the present invention has the property of acting only on cancer cells without affecting normal cells (selective cell killing activity, that is,
Antitumor activity).

The antitumor activity of the protein according to the invention is
It can be confirmed by using a cell obtained by transforming a mouse normal cell (for example, fetal fibroblast) with a tumor virus (for example, polyoma virus) and measuring the growth inhibitory activity on the tumor cell.

The measurement of the antitumor activity is carried out by a generally used method, for example, a method using a mouse transplanted with a tumor (Ki
kuo Nomoto, Chikao Yoshikumi, Kenichi Matunaga, Takay
oshiFujii and Kenji Takeya “Restoration of Antibo
dy-forming Capacities byPS-K in Tumor-bearing Mic
e "Gann 66 by (1975) 365-374) may be performed in in vivo. However, by measuring the cell proliferation suppressing effect on the invitro using animal cells transformed with tumor viruses, facilities required for research This is a significant advantage over the in vivo method in terms of cost, time, and the like.
A preferred example of a method for measuring the cell growth inhibitory effect in vitro is described in Example 2.

According to the above-described measuring method, the protein according to the present invention has a final concentration of 11 n as shown in FIG.
The cell growth inhibitory effect was already shown at g / ml. Also, LD
₅₀ (half inhibitory concentration) is 22-59 ng / ml,
At 88 ng / ml, both cells almost die. Even more surprisingly, at a concentration of 44 ng / ml, the viability of the transformed cells was about 15%, while the viability of the normal cells was about 60%. This indicates that the protein according to the present invention has excellent antitumor activity with low side effects.

The antitumor activity of the protein according to the invention is 6
When the temperature exceeds 0 ° C., the temperature is remarkably reduced. In addition, the protein itself precipitates when the temperature exceeds 60 ° C. The antitumor activity of the protein according to the invention is also stable over a wide range of pH 3-12.

The protein according to the invention has the property of salting out with 60% ammonium sulfate, preferably 40% ammonium sulfate.

The protein according to the present invention has an estimated molecular weight of 150 to 200 kDa by gel filtration,
The estimated molecular weight by gel filtration is about 200 kDa.
The estimated molecular weight by SDS-PAGE was 62 kDa when treated at 98 ° C, and 13 kDa when treated at 37 ° C.
5 kDa and 62 kDa.

The protein according to the present invention was prepared in Example 3
It has the amino acid composition described in (2). The protein according to the invention is relatively rich in acidic amino acids and is considered to be an acidic protein. Further, the protein according to the present invention has a sugar chain.

As described below, the protein according to the present invention is considered to exist in the form of a dimer (for example, dimer or tetramer).

The basic subunit is a protein having a molecular weight of 62 kDa, and this protein is presumed to have the amino acid sequence of SEQ ID NO: 1.

The protein according to the present invention can be produced, for example, as follows, from the fruiting body of Amanita mushroom.

First, raw or frozen ash mushrooms ( P
leurotus cornucopiae ) is finely divided with scissors or the like, and a suitable amount (preferably about twice as much) of water or a buffer solution containing an inorganic acid salt or an organic acid salt (for example,
0.15 M sodium chloride and 0.01 M Tris-HCl buffer (pH 7.4) are added, and the mixture is crushed with a homogenizer or the like for an appropriate time (for example, about several minutes) and stirred. It is preferable that the fruit body of Tamamotake to be extracted is finely crushed in consideration of the extraction efficiency. Also,
In the case of a frozen product, it is preferable to use a product after thawing. The Tamago mushroom used may be either a native one or a commercially available one.

The extract is centrifuged at a low temperature (preferably about 4 ° C.) (typically at 5,000 to 10,000 rpm).
m. , 10 to 45 minutes) to obtain a supernatant. Further, an equal amount of water or a buffer solution is added to the precipitate, followed by re-extraction to obtain a supernatant. The crude supernatant can be obtained by combining the extraction supernatants twice (or more times) (see FIG. 2).
As another method, extraction can be performed once with more than 2 times (preferably 4 times or more) of water or buffer.

Next, ammonium sulfate is added to the crude extract to carry out salting out (60% saturation concentration, preferably 40% saturation concentration), and centrifugation (usually at 5,000 to 10,000 rpm,
The precipitate obtained under the conditions of 10 to 45 minutes) is desalted by dialysis,
An ammonium sulfate salt out product can be obtained. As a method other than salting out, a high molecular fraction can be obtained by ultrafiltration. This ammonium sulfate precipitate or ultrafiltration polymer fraction is
(1) contacting with an anion exchange resin to perform an adsorption treatment, and then elute the protein from the column with an eluent;
(2) a step of contacting with a resin for hydrophobic chromatography to carry out an adsorption treatment, followed by eluting the protein from the column with an eluent, and (3) a step of purifying the protein by a molecular weight fractionation treatment with a gel filtration column. An antitumor protein can be obtained by a purification step in which various types of purification steps are combined in an arbitrary order. The antitumor protein thus obtained is as described above in (1) to
It has the property of (5).

When the protein according to the present invention is used as an antitumor agent, the active fraction of the final chromatograph is usually used as an active substance, but if necessary, the active fraction before this final purification step may be used. Further, a recombinant protein as described below may be used. According to the present invention, there is also provided a recombinant protein comprising the amino acid sequence of SEQ ID NO: 1. This recombinant protein also has antitumor activity (Example 5).

[0025] The recombinant protein according to the invention may have one or more additions, insertions, substitutions and / or deletions in the amino acid sequence of SEQ ID NO: 1. The term “addition, insertion, substitution, and deletion” as used herein refers to those that do not impair the antitumor activity of the protein consisting of the amino acid sequence of SEQ ID NO: 1. The number of additions, insertions, substitutions, and deletions is not particularly limited as long as the antitumor activity is not impaired.

Examples of the deletion include deletion of one or more amino acid residues selected from amino acid residues 1 to 46 in the amino acid sequence of SEQ ID NO: 1. This deletion is performed by deleting consecutive amino acid residues of amino acids 1 to X (X represents an integer of 1 to 46) in the amino acid sequence of SEQ ID NO: 1 (for example, 1 to X).
Deletion of the 46th amino acid residue). Additions, insertions, substitutions, and deletions in amino acid sequences can be made, for example, by using the Molecular Cloning A Laboratory man.
ual), second edition, Cold Spring HarborLaborator
y Press, Vol. 2, Chap. 15 (1989); Bostein, D. et al., Sc
ience, 229: 1193 (1985); Craik, CS, Biotechniques, 3:
12 (1985); Itakura, K. et.al., Annu. Rev. Biochem. 5
3: 323 (1984); Shortle, D. et al., Annu. Rev. Genet.
15: 265 (1981); Smith, M. Annu, Rev. Genet. 19: 423 (19
85). In the present invention, the term “protein having antitumor activity” refers to a protein that has been evaluated by those skilled in the art as having antitumor activity. For example, when a protein is tested under the same conditions as in Example 2, the protein has antitumor activity. Shall mean the protein that is evaluated.

The recombinant protein is, for example, its cD
It can be obtained by expressing the NA sequence (DNA sequence of SEQ ID NO: 2) in a host (for example, Escherichia coli) according to a conventional method (Example 5). cDNA uses an antibody against a protein having antitumor activity as a probe,
It can be obtained by screening a cDNA library derived from Agaricus blazei (Example 4).

According to [0028] nucleotide sequences present invention, the nucleotide sequence encoding the protein according to the invention is provided. A typical sequence of this nucleotide sequence has part or all of the DNA sequence of SEQ ID NO: 2.

The DNA sequence of SEQ ID NO: 2 was obtained from a cDNA library derived from Aspergillus niger as described above. This DNA sequence contains the open reading frame of the protein, the open reading frames starting from ATG 1-3 and 1900-19.
It ends with 02 TAA (stop codon).

Given the amino acid sequence of the protein according to the present invention, the nucleotide sequence encoding it can be easily determined, and various nucleotide sequences encoding the amino acid sequence shown in SEQ ID NO: 1 can be selected.

Accordingly, the nucleotide sequence encoding the protein according to the present invention is defined as the DNA of SEQ ID NO: 2.
A DNA sequence encoding the same amino acid, in addition to part or all of the sequence,
A sequence having a sequence is also meant, and an RNA sequence corresponding thereto is also included.

[0032] The nucleotide sequence according to the present invention may be of natural origin or may be totally synthetic.
In addition, a product synthesized using a part of a product derived from a natural product may be used. The nucleotide sequence can be obtained from a chromosome library or a cDNA library by a method commonly used in the field of genetic engineering, for example, a method of screening using an appropriate probe prepared based on partial amino acid sequence information, and the like. Can be. The nucleotide sequence according to the present invention may be, for example,
It can be obtained from a DNA library by using an oligonucleotide corresponding to the amino acid sequence of positions 48 to 97 of SEQ ID NO: 1 as a probe in screening.

When the nucleotide sequence is derived from a natural product, its origin is not particularly limited, and it may be derived from Tamamotake or from other sources.

According to vectors and transformation invention, a vector comprising a nucleotide sequence according to the invention described above, the vector is in replicable state in a host cell, and in a state capable of expressing a protein whose nucleotide sequence encodes Is provided. Further, according to the present invention, there is provided a host cell transformed with the nucleotide sequence encoding the vector or the antitumor protein. The host-vector system is not particularly limited,
In addition, a fusion protein expression system with another protein can be used.

As a fusion protein expression system, β-galactosidase, glutathione-S-transferase,
Fusion proteins such as luciferase;

As the vector, a plasmid vector (for example, pBluescript (registered trademark) SK (-), pBluescript
(Registered trademark) SK (+), pGEX-4T, pGEX-5
T, pRIT2T, pBPV, and pSVK3 (Pharmacia, etc.) and ZAP Express, p
YEUra3, pMAM, and pOG (Toyobo, etc.), viral vectors (for example, retrovirus vectors, adenovirus vectors), and liposome vectors (for example, cationic liposome vectors).

In order to express a desired protein by actually introducing the vector into a host cell, the vector according to the present invention may contain, in addition to the nucleotide sequence according to the present invention, a sequence controlling its expression (for example, a promoter). Sequences, terminator sequences, enhancer sequences) and genetic markers for selecting host cells (eg, neomycin resistance gene, kanamycin resistance gene), and the like. The vector may also contain the nucleotide sequence according to the present invention in a repeated form (eg, in tandem). These may be introduced into a vector according to a conventional method,
As a method for transforming a host cell with this vector, a method commonly used in this field can be used.

As the procedure and method for constructing a vector according to the present invention, those commonly used in the field of genetic engineering can be used.

As the host cell, for example, Escherichia coli (eg, SOLR (Stratagene), JM1
09 (Toyobo), XL1-Blue MRF '(ST
RATAGENE), DH5α (Toyobo), BL2
1 (DE3) (Novagen)), yeast (for example,
YRG-2 strain), Bacillus subtilis, insect cells, animal cells (for example, CHO cells, COS cells, human keratinocytes, C
OP-5, C127, mouse 3T3 cells, FR3T3,
HB101 etc.) and plant cells.

The transformed host cells are cultured in an appropriate medium, and the above-mentioned protein according to the present invention can be obtained from the culture. Thus, according to another aspect of the present invention, there is provided a method for producing a protein according to the present invention. By this method, a large amount of an antitumor protein can be produced.

Uses / Pharmaceutical Compositions The proteins according to the invention have antitumor activity. Thus, according to the present invention there is provided a pharmaceutical composition comprising a protein according to the present invention. Pharmaceutical compositions according to the present invention include cervical cancer, endometrial cancer, epithelial cancer, stomach cancer, esophageal cancer,
Treatment of intestinal cancer, rectal cancer, pharyngeal cancer, oral cancer, maxillary cancer, and various cancers caused by abnormal expression of the tumor suppressor genes p53 and pBR (eg, skin cancer, lung cancer, liver cancer, kidney cancer, breast cancer, etc.) It can be used as an agent.

The pharmaceutical composition according to the present invention can also be administered orally or parenterally (eg, intramuscular, intravenous, subcutaneous, rectal, transdermal, nasal, sublingual, etc.). It is used in humans and non-human animals in various dosage forms suitable for oral or parenteral administration as medicaments. Stomach cancer, esophageal cancer, intestinal cancer, lung cancer, kidney cancer, liver cancer,
Oral administration for breast cancer, administration of suppositories for rectal cancer, oral administration of troche, etc. for pharyngeal, oral and maxillary cancer, or administration of direct application, epithelial cancer and skin cancer However, it is considered that administration by direct application or administration such as injection for other cancers is an effective administration method, but is not limited thereto.

The protein according to the present invention may be used in the form of tablets, capsules, granules, powders, pills, fine granules, lozenges, orally; Formulations, oily suppositories, water-soluble suppositories and the like. These various formulations include excipients (eg, fillers and fillers), adjuvants (eg,
Binders, wetting agents, disintegrants, surfactants, lubricants, dispersants, buffers, and solubilizing agents), and additives (eg, preservatives, flavoring agents, soothing agents, stabilizers, Coloring agents and sweeteners) can be used in the usual manner.

The dosage for various treatments can be appropriately determined in consideration of the usage, the age, sex, degree of symptoms, etc. of the patient.

Antibodies According to the present invention there are provided antibodies against the proteins according to the present invention. Antibodies according to the present invention include polyclonal and monoclonal antibodies.

The antibodies according to the present invention can be produced by conventional techniques. For example, a protein described in SEQ ID NO: 1 can be obtained by adding an arbitrary carrier (eg, Freund's complete and incomplete adjuvant) together with an animal body (eg,
Rabbits, rats and mice), and after a certain period of time,
It can be obtained by purifying the serum of the animal.

The specific reaction of the polyclonal antibody (ie, immune reaction) is one indicator of the presence of an antitumor protein. Therefore, the antibody according to the present invention is useful as an antibody for antitumor protein purification or screening.

[0048]

The present invention will be described in detail with reference to the following Examples, but it should not be construed that the invention is limited thereto.

Example 1 Preparation of Test Substance Derived from Mushroom Pleurotus cornucopiae and matsutake ( Tricholoma ) as a commercially available edible mushroom
matsutake ), Enokitake ( Flammulina velutipes )
, Hon Shimeji ( Lyophyllum shimeji ), Shimeji ( Ple
urotus ostreatus ), shiitake mushroom ( Lentinus edodes )
, Abalone mushroom ( Pleurotus ostreatus ), Oyster mushroom (
Pleurotus ostreatus ), white mushrooms (
Agaricusbisporus ), Brown Mushroom ( Agaric )
us bisporus ), nameko ( Pholiota nameko ) and maitake ( Grifola frondosa ) were screened for antitumor activity.

First, the raw or frozen fruit body of each mushroom (after thawing in the case of a frozen one) is finely ground with scissors, and twice the amount of 0.15M sodium chloride containing 0.15M sodium chloride.
A 01 M Tris-HCl buffer (pH 7.4) was added, crushed with a homogenizer, and stirred. This crushed material is kept under low temperature (4
° C) and centrifugation (8,000 rpm, 30 minutes)
Thus, a crude extract was obtained. Ammonium sulfate was dissolved in each crude extract to 90% saturation and centrifuged (8,000 rpm,
30 minutes) to collect the precipitate, and dialyzing membrane Dialysis
Membrane, Size 36 (manufactured by Wako Pure Chemical Industries, Ltd.
The resulting supernatant was desalted and centrifuged by dialysis using a fractionated molecular weight of 12,000 to 14,000) to obtain an ammonium sulfate precipitate (see FIG. 1).

Example 2 Antitumor Activity Test Mouse Fetal Fibroblast BALB / 3T3 Clone A31
(RIKEN GeneBank (RIKEN)) and SV-T obtained by transforming the same with polyoma virus SV40.
2 (RIKEN GeneBank) in a medium (Dulbecco's modified Eagle medium) containing 10% serum (calf serum)
Seeded at ⁴ cells / ml, 24 hours at 37 ° C, 5
Cultured under% CO ₂ . Here, the PBS solution of each sample prepared in Example 1 was added, and the cells were further cultured for 48 hours, and the number of cells at this time was measured by the MTT method.

The MTT method was performed as follows. First,
Four hours before the measurement, phosphate buffered saline (PBS solution) of MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromine) was added to the cells, and the cells were further added for 4 hours. Cultured for hours. Then, after removing the culture supernatant, 100 μl of hydrochloric acid (0.04 N) isopropanol and 20 μl of a 3% SDS aqueous solution were added per well of a 96-well microplate to dissolve formazan generated by the action of mitochondria of living cells.
The solution was measured for absorbance at 595 nm (control 655 nm) (Tim Mosmann "Rapid Colorimetric Assay for Cell").
ular Growth and Survival: Applicationto Proliferat
ion and Cytotoxicity Assays ”Journal of Immunolog
ical Methods 65 (1983) 55-63; Lora M. Green, Jeanne L.
Reade and Carl F.Ware “RapidColormetric Assay for
Cell Viability: Application to the Quantitation of
Cytotoxic and Growth Inhibitory Limphokines ”Jou
rnal of Immunological Methods 70 (1984) 257-268).

The number of cells measured by the MTT method was expressed as a percentage of the number of control cells (see FIG. 3).

Each crude extract and the ammonium sulfate precipitate obtained in Example 1 were subjected to an antitumor activity test. The results were as shown in Tables 1 and 2. The crude extract and ammonium sulfate precipitate of the fruit body of Pleurotus cornucopia showed remarkably high antitumor activity (the effect of suppressing the growth of tumor cells).

[0055]

[Table 2]

[0056]

[Table 3] Example 3: Purification of anti-tumor protein from Pleurotus cornucopia
And antitumor test (1) Purification of antitumor protein After thawing 2,000 g of frozen fruit body of Agaricus blazei, crush it with scissors, and add PM as a protease inhibitor.
SF1 mM, Pepstatin A 1 μg / ml, EDTA1
0.01 containing 0.15 M sodium chloride plus mM
M Tris-HCl buffer (pH 7.4) was doubled (4,000
0 ml) and crushed with a homogenizer for several minutes. This crushed material is centrifuged at a low temperature (4 ° C.) (8,000 rpm).
p. m. , 30 minutes) to obtain a supernatant (5,190 ml) and a precipitate. An equal amount of the same buffer (2,000 ml) was added to the precipitate, and the mixture was re-extracted, and centrifuged to obtain a supernatant (1,925 ml). Combine the two supernatants and extract crude (7,115 ml)
I got Dissolve ammonium sulfate in this so that it is 40% saturated,
A precipitate was obtained by centrifugation (see FIG. 2). Then 40%
Ammonium sulfate was dissolved in the supernatant so as to be 60% saturated, and a precipitate was similarly obtained. A precipitate was also obtained at 80% saturation. Precipitate at each saturation concentration is passed through a dialysis membrane DialysisMemb
Rane, Size 36 (manufactured by Wako Pure Chemical Industries, Ltd., molecular weight cut off: 12,000 to 14,000), to which PMSF 0.1 mM, pepstatin A 0.1 μg / ml, and EDTA 0.1 mM were added as protease inhibitors 5
Desalting was performed by dialysis against a 0 mM Tris-HCl buffer to obtain each centrifuged supernatant, and the activities were compared. As a result, it was confirmed that most of the activity in the buffer extract was transferred to the 40% precipitated fraction (data omitted). The protease was added at the same concentration to the anion exchange chromatography, hydrophobic chromatography, and eluate for gel filtration.

The supernatant obtained by centrifugation was subjected to TSKgel DEAE-
The protein was applied to an anion exchange chromatography column using Toyopearl 650M (manufactured by Tosoh Corporation), and after adsorption and washing, the protein was eluted with a linear concentration gradient of NaCl (FIG. 4). The active fraction was observed at a NaCl concentration of around 0.2M.

Next, the active fraction was subjected to TSKgel Phe
The protein was applied to a hydrophobic chromatography column using nyl-Toyopearl 650M (manufactured by Tosoh Corporation), and after adsorption and washing, the protein was eluted with a linear concentration gradient of ammonium sulfate (FIG. 5).
The active fraction was found in the fraction eluted with 0 M ammonium sulfate.

Further, after the protein was salted out with ammonium sulfate, it was redissolved in water, concentrated, and separated by Sepharose.
CL-6B (manufactured by Alsham Pharmacia Biotech)
Was used for gel filtration. As a result, antitumor activity was observed at the main peak (FIG. 6).

Table 3 shows the data of the active fraction for each purification step.

[0061]

[Table 4]

(2) Characterization of antitumor protein Molecular weight Sepharose CL-6B was used to measure the activity of each of the gel filtration fractions. Fractions 39 to 47 showed activity. Further, blue dextran, thyroglobulin, alcohol dehydrogenase, bovine serum albumin, ovalbumin, and ribonuclease A were used as standard substances for molecular weight estimation.
From this, the molecular weight of the gel filtration active fraction (hereinafter referred to as “anti-tumor protein”) by the gel filtration method was 150 to 20.
It was estimated to be 0 kDa.

The purified sample antitumor protein was
After treating at 37 ° C. or 37 ° C., the result of SDS-polyacrylamide electrophoresis was as shown in FIG.
As is clear from the photograph, bands of 135 kDa and 62 kDa were detected in the sample treated at 37 ° C.
A single band (62 k
Da) was detected. Therefore, it is considered that the antitumor protein has a polypeptide having a molecular weight of 62 kDa as a basic subunit and exists in the form of a complex (eg, dimer or tetramer). In addition, HPLC gel filtration using TSKgel G3000SW _XL (manufactured by Tosoh Corporation) was performed for the antitumor protein. FIG. 8 shows the elution pattern. When the activity was measured in two fractions, a strong activity was observed in fraction 1. Thyroglobulin, alcohol dehydrogenase, bovine serum albumin, ovalbumin and ribonuclease A were used as standard proteins for estimating the molecular weight. From this, the molecular weight of the antitumor protein by HPLC was estimated to be about 200 kDa.

Amino acid composition ratio The results of the amino acid composition analysis of the present antitumor protein purified to homogeneity as described above are as follows. The amino acid composition analysis was performed by using a sodium citrate buffer (pH 3.200 and 10.000) and a Na ⁺ type strongly acidic cation exchange resin column (Shimpak ISC-07 / S1504 Na, 6.
0x1000mm, Shimadzu Corporation) and post column OPA
The concentration gradient program for hydrolysis of the amino acid analysis system manufactured by Shimadzu Corporation by the derivatization method was partially modified, and measurement was performed by the one-point absolute calibration curve external standard method. Amino acid standard solutions include:
The measurement was performed using an amino acid mixed standard solution (Wako Pure Chemical Industries, Ltd., H type).

[0065]

[Table 5]

Ultraviolet Absorption Spectrum When an ultraviolet absorption spectrum of the antitumor protein was measured, a maximum absorption was observed at a wavelength around 277 nm (FIG. 9).

Effect of heat The antitumor proteins were reduced to 4, 10, 20, 30, 40, 5
0, 60, 65, 70, 75, 80, 90 and 100 ° C
At each temperature for 10 minutes, and the cell growth inhibitory activity of each temperature-treated sample was measured. As shown in FIG. 10, the activity of the protein was stable up to 60 ° C., and when it exceeded 60 ° C., the activity was significantly reduced.

Effect of pH Antitumor proteins were treated with 50 mM sodium acetate-hydrochloric acid pH
1,2,50mM citric acid-sodium hydrogen phosphate p
H3, 4, 5, 6, 7, 50 mM Tris-HCl pH 8,
9, 50 mM glycine / sodium chloride-sodium hydroxide were added to the respective buffer solutions of pH 10, 11, 12, and 12.5, and left at low temperature (4 ° C.) for 24 hours. PH of each mixture
Was neutralized with 500, 50, and 5 mM hydrochloric acid and sodium hydroxide (in the range of pH 7.2 to 7.4), and the volume was adjusted to a fixed volume with PBS. As shown in FIG. 11, the activity of the protein is p
Stable at H3-12 and deactivated at a pH of 2 or less.

Amino acid sequence Using an antitumor protein as a sample, SDS was prepared using SDS-containing buffer containing SDS and 2-mercaptoethanol.
After the formation, the gel was subjected to SDS-polyacrylamide electrophoresis.
After the electrophoresis, the proteins in the gel were transferred onto a PVDF (polyvinylidene fluoride) membrane by blotting. PVDF membrane is coated with CBB (Coomassie Brilliant Blue)
After staining and drying, a band portion of the target protein (portion having a molecular weight of about 62 kDa and about 135 kDa) was cut out and supplied to an amino acid sequencer (Hewlett-Packard). The analysis result of the 62 kDa portion is shown in FIG.
2 as described. Reliable data is shown in the upper part of FIG. 12 and in SEQ ID NO: 3. It was also confirmed that the N-terminal sequences of the two bands (62 kDa and 135 kDa) were the same. Thus, an anti-tumor protein is a complex of the basic peptide (62 kDa) (eg,
Dimer or tetramer).

Sugar Chain The antitumor protein was subjected to PAS staining to examine the presence of the sugar chain. As a result, the presence of a sugar chain was confirmed in the protein of the present invention (data omitted).

(3) Antitumor Activity Test Using the activity measurement method described in Example 2, the relationship between the concentration of the antitumor substance prepared in Example 3 and the cell growth inhibitory rate was examined. As a result, as shown in FIG.
1 already showed a cell growth inhibitory effect. The LD ₅₀ (half inhibitory concentration) is about 22-59 ng / ml and about 88
At ng / ml, both cells almost died.

In particular, when the concentration was 44 ng / ml, the viability of the transformed cells was about 15%, whereas the viability of the normal cells was 60%. LD ₅₀ values for mice normal cells of the purified protein (BALB / A31) is 59ng
/ Ml, the concentration that killed half of the transformed cells (SV-T2) was 22 ng / ml.

Further, its action characteristics were effective not only for mice but also for human cancer cells and the like. For the HeLa LD ₅₀ value is a human uterine cancer-derived cells about 45
ng / ml human fetal lung cells were transformed with SV40
46 ng / ml for WI38 VA13 subline 2RA, 47 ng / ml for mouse leukemia cell P388D1
And 48 ng / ml for human acute lymphocytic leukemia cell MOLT-4. The relative lethal activity was as shown in Table 4.

[0074]

[Table 6]

(4) Preparation of Antibody The protein prepared in (1) was added to a buffer (50 mM Tris-HCl) together with Freund's complete adjuvant.
(PH 7.5, 0.1 mM PMSF, 0.1 μg / m
l pepstatin A, 0.1 mM EDTA)
suspended at a concentration of 0.2 mg / ml.
Was injected into rabbits. A sample (0.2 g of protein) mixed with Freund's incomplete adjuvant was boost-immunized after 2, 5 and 7 weeks. Eleven weeks after the first immunization, whole blood was collected to obtain antisera.

Example 4 cDNA Cloning 6.2 g of fresh fruit body of Agaricus blazei (commercially available) was used as a sample, and total RNA was extracted by the SDS / phenol method.
After the sample was crushed in a mortar, denatured proteins and DNA were separated and removed. The obtained RNA was dissolved in TE buffer,
As a result of measuring the absorption at nm and 280 nm, 7.1 m was obtained.
g of total RNA was obtained. Of these, 500 μg of total RN
A as a sample, Poly (A) ⁺ -mRNA was extracted from the nucleic acid sample using Oligo-dT Latex (Oligo-d
T particles, specifically Oligotex®-dT
30) mR of antitumor protein using (Takara)
NA was purified. As a result, 13 μg of mRNA was obtained. Based on 5.0 μg of mRNA, ZAP-cDNA (trade name) Synthesis Kit (STRATATAG)
CDNA was synthesized using ENE (sold by Toyobo). After cDNA synthesis, Uni-ZAP (trade name) X
ΛZAP (registered trademark) II in R vector Kit
/ Eco RI / Xho I / CIAP treated vector. GIGAPACK (registered trademark) III
Gold Packing Extract (STR
ΛZAP manufactured by ATAGENE and sold by Toyobo
A phage library was constructed by in vitro packaging into (registered trademark) II (phage vector).

Using the antibody obtained by the method described in Example 3 (4) as a probe, phage having an antitumor protein gene was subjected to antibody screening from a phage library, and 6 positive phages (clone No. 1) were screened. ,
3, 4, 5, 6, 7) were obtained. Specifically, the procedure was performed as follows.

The titer of the phage library was measured. Dilute phage with SM buffer, dilute 2-20 μl
1 (phage number: about 2,000 to 20,000) was added with 200 μl of a suspension of Escherichia coli XL1-Blue strain, mixed, and cultured at 37 ° C. for 15 minutes with shaking. 3.0 ml NZYM Top Agar kept at 45 ° C
(0.7%), 15 μl IPTG (0.5 M, aqueous solution), 50 μl X-GAL (250 mg / ml, DM
F solution), and after mixing, spread evenly on a plate of 135 mm diameter NZYM medium. The mixture was left standing at room temperature for about 10 minutes to solidify, and cultured at 37 ° C. until the size of the plaque became about 1 mm. The clear plaque was counted, and the titer was 2.5 × 10 ⁵ pfu.

A primary screening of positive phage was performed. Escherichia coli XL-1-Blue was added to 100 μl of the phage solution.
The suspension of the strain was added in an amount of 600 μl, and cultured at 37 ° C. for 15 minutes. NZYM Top Aga kept at 45 ° C
After adding 8 ml of r and mixing, the mixture was spread evenly on a plate of NZYM medium. Allow to set for several minutes at room temperature, 4
After culturing at 2 ° C. for 3 to 4 hours, a 130-mm-diameter nitrocellulose membrane (Schleicher & Schuell) impregnated with 10 mM IPTG and dried was overlaid on the plate, and incubated at 37 ° C. for 3 hours. After the plate was cooled at 4 ° C. for 1 hour or more, the nitrocellulose membrane was peeled off from the plate, and TB containing 3% skim milk was added.
The plate was shaken with an ST buffer for 1 hour or more, and washed several times with TBS-T for washing. The antibody was immersed in the TBS-T diluent described in Example 3 (4) and treated at room temperature for 1 to 2 hours.
The plate was blocked with a TBS-T buffer containing 3% skim milk and washed several times with TBS-T. 100 with TBS-T
Secondary antibody solution diluted 0-fold (Promega, Ant
i-Rabbit IgG (Fc), AP Conju
gate) and treated at room temperature for 1-2 hours. TBS
-Wash several times with -T, alkaline phosphatase (AP)
Washed once with buffer. Chromogenic substrate NBT (= Nitro
blueetrazolium, 75 mg / ml, A
1/400 volume of P buffer) and BCIP (= 5-bro
mo-4-chloro-3-indolphosph
ate, 50 mg / ml, 1/400 liquid volume of the AP buffer) was added, and phage showing a positive reaction was detected. Thereafter, the nitrocellulose membrane was washed with distilled water and dried.

The positive phages obtained in the primary screening were collected, suspended in 500 μl of SM buffer, and used as a sample. As a result of the secondary screening performed in the same manner as described above, six positive phages were obtained (No.
1, 3, 4, 5, 6, 7) No. 1 positive phage, Z
AP-cDNA Synthesis Kit (STR
ExAss made by ATAGEN, sold by Toyobo
ist (trade name) helper pharge, using E. coli XL-1 Blue strain, invivo Exc
Phazimid was obtained by the ision method.

This phagemid and ZAP-cDNA Sy
nthesis Kit (manufactured by STRATAGENE,
Distributor: Toyobo) E. coli SOLR strain (STRATA)
(GENE) to obtain a transformant.

Using this transformant as a sample, Wizard
(Registered trademark) Plus SV Minipreps
(Promega, sales agency: Funakoshi) using
Plasmid DNA was prepared.

This plasmid DNA was ligated with EcoRI and
After treatment with Kpn I, agarose gel electrophoresis was performed.
As a result, the size of the insert DNA was about 2000 bp.

The entire nucleotide sequence of the insert DNA region of the plasmid DNA obtained above was compared with Dye P
It was determined by the primer method and the Dye Terminator method (SEQ ID NO: 2). Reagents include ABI PRIS
M (trade name) Dye Primer Cycle Se
quenching FS Ready Reactio
n Kit and ABI PRISM (trademark) Big D
ye (trade name) Terminator Cycle S
equaling Ready Reaction
Kit (PerkinElmer) was used. The DNA sequencer is ABI PRISM (trade name) 377 DN
A Sequencer, 310 Genetic A
Nalyzer (PerkinElmer) was used.

The amino acid sequence of the antitumor protein was obtained from SEQ ID NO: 2 (SEQ ID NO: 1). The molecular weight estimated from the amino acid sequence was about 70.3 kDa.

The sequence (SEQ ID NO: 3) of the peptide fragment obtained by the amino acid sequence of (2) in Example 3 partially coincided with the amino acid sequence of positions 47 to 96 of SEQ ID NO: 1.

The amino acid composition ratio of the amino acid sequence of SEQ ID NO: 1 was compared with the result of amino acid composition analysis of the purified protein. The results were as shown in Table 5.

[0088]

[Table 7] Asparagine and glutamine are converted to aspartic acid and glutamic acid, respectively, by hydrolysis in amino acid composition analysis. Therefore, there is no data on the composition ratio of asparagine and glutamine in the purified protein. Therefore, in the amino acid composition ratio obtained from the deduced amino acid sequence, the value of asparagine + aspartic acid and the value of glutamine + glutamic acid were compared with the amino acid composition ratio of aspartic acid and glutamic acid of the purified protein, respectively.

Further, tryptophan cannot be analyzed by analyzing the composition of amino acids hydrolyzed with hydrochloric acid. Therefore, there is no data on the amino acid composition ratio of tryptophan in the purified protein.

As shown in Table 5, the results of the amino acid composition analysis of the purified protein and the amino acid composition ratio of the deduced amino acid sequence were almost the same. This indicates that the purified protein was obtained with high purity, and that the obtained cDNA sequence encodes the antitumor protein according to the present invention.

DNA sequence of SEQ ID NO: 2 and SEQ ID NO: 1
For each amino acid sequence.
The database is DDBJ and Gen for DNA sequences.
bank and EMBL were used, and Swiss-Prot, PIR, GenPept and PDB were used for amino acid sequences, respectively. As a result, no amino acid sequence or DNA sequence having high homology to the proteins and genes of the present application was found.

Example 5 Production of recombinant protein (1) Selection of vector and design of insert DNA pET-20b was used as a vector for expressing a recombinant gene.
(+) (Novagen) was selected. In preparing the expression vector, DNA to which a restriction enzyme site not present in the cDNA of the antitumor protein according to the present invention was added as an adapter was synthesized, and this was incorporated into the vector. Restriction enzyme sites are Nde I and Ec
o RI. Primers using these restriction enzyme sites as adapters are as shown in FIG. Using these, a DNA fragment (total sequence (Met to (stop codon), 1,902 bp) and a DNA fragment (N-terminal deletion sequence (Lys, Leu, Pro ... (stop codon), 138 bp from the fragment (46 amino acid residues) (2) Expression test method and results DNA fragments were synthesized by PCR using the primers shown in Fig. 14. Each primer and template DNA dissolved in TE buffer CDNA obtained in Example 4 (plasmid D prepared in Example 4)
NA) using TaKaRa LA PCR (trade name)
Kit Ver. PCR using 2 (TAKARA)
DNA fragments were synthesized by the method.

DNA fragment and vector p
ET-20b (+) was treated with Nde I and Eco RI. The vector pET-20b (+) was transformed into Bacter
ial Alkaline Phosphatases
(Toyobo Co., Ltd.). Thereafter, agarose gel electrophoresis was carried out, a gel portion where each DNA was present was cut out, extracted and purified. pET-20b (+) and D
The NA fragment and each were mixed at 16 ° C.
DNA fragment and p
Ligation with ET-20b (+).

The ligated plasmid DNA was used to transform E. coli DH5α strain. LB / Amp plate (LUURIA BROTH, 25 g / liter,
The bacterial cell solution was applied to SIGMA / ampicillin (50 μg / ml), cultured at 37 ° C., and the colony-forming bacterial cells were suspended in a buffer. Insert DNA in suspension
After amplification by the CR method, the resultant was subjected to agarose gel electrophoresis to identify cells having an insert DNA, ie, transformants. In order to confirm whether or not the synthesized insert DNA had a mutation, plasmid DNA prepared from each transformant was amplified by PCR. Each plasmid DN
A was subjected to agarose gel electrophoresis and the insert DNA
Was confirmed from the electrophoresis pattern as to whether it was a transformant having a plasmid DNA containing the entire length of the plasmid.

For the obtained transformant, the entire sequence of the insert portion in the plasmid DNA was determined using Dye-T (using the primers used in the cDNA sequencing).
It was confirmed by the emitter method. The reagent is AB
I PRISM (trade name) dRhodamine Te
rminer Cycle Sequencing
FS Ready Reaction Kit (PerkinElmer) and DNA sequencer ABI
PRISM (trade name) 377 DNA Sequence
r (Perkin Elmer) was used. As a result, no change in the nucleotide sequence was observed in the synthesized DNA fragment and the synthesized DNA fragment, and the cDN of the antitumor protein of the present invention
A plasmid DNA which completely matched A and had these as insert DNAs was obtained.

Using these plasmid DNAs, Escherichia coli strain BL21 (DE3) was transformed. The bacterial cell fluid of the transformant was spread on an LB / Amp plate and cultured at 37 ° C. to prepare a plasmid DNA from the bacterial cells that formed colonies. As a result of agarose gel electrophoresis, it was confirmed that there was no change in the size of the plasmid DNA.

Using the transformant thus obtained, an expression test was performed as follows. First, LB / Am
The colony on the p-plate was scraped with a platinum loop and LB / A
After suspending in 2.0 ml of mp liquid medium, the cells were cultured with shaking at 37 ° C. overnight. 200 μl of the culture solution in which the cells were grown was added to LB /
The mixture was mixed with 20 ml of Amp liquid medium, shake-cultured at 37 ° C. for about 2 hours, and cultured until the absorbance at 600 nm became 0.2 to 0.6. IPTG (Isopropyl-β-D
-Thiogalactopyranoside) was added at 0.4 mM to induce expression. After culturing with shaking at 18 ° C. overnight, the amount of bacterial cells was measured from the absorption at 600 nm, and the amount of the extraction buffer was determined. Centrifuge the cells (3,
500r. p. m. , 4 ° C, 5 minutes).

The extraction buffer contains 25 mM Tris-H.
Cl, 150 mM NaCl, 10 μM EDTA, 1
0 μM PMSF, 10 ng / ml pepstatin A,
Each cell was suspended in a buffer using 10 ng / ml leupeptin. Ultrasonic crusher (BRANSON ULTR)
ASONICS, Model450 Sonifie
r (registered trademark)), the output: 5.0, D
The cells were disrupted while cooling on ice under the conditions of a Util cycle: 80% and an extraction time of 4 minutes (1 or 2 times). The soluble fraction and the insoluble fraction were collected by centrifugation (7,000 rpm, 4 ° C., 10 minutes).

The obtained soluble and insoluble fractions were
After heat treatment (3 minutes in boiling water) or non-heat treatment using an SDS-containing buffer containing SDS and DTT,
It was subjected to DS-polyacrylamide gel electrophoresis. Furthermore, the proteins in the gel were converted to PVDF (Polyvini
den fluoride) Example 3
It was subjected to Western blot analysis using the antiserum obtained by the method described in (4). The results were as shown in FIG. DNA fragments and expressed proteins were identified in the soluble and insoluble fractions.
The expressed protein of the DNA fragment had almost the same molecular weight as the monomer of the protein purified from the fruit body of Pleurotus cornucopiae, and had a size of 60 to 65 kDa.
The molecular weight of the expressed protein of the DNA fragment was 65-70 kDa as deduced from the DNA sequence of the antitumor protein according to the invention. A high molecular weight component presumed to be a dimer was also detected, and there was also a reaction in Western blot analysis, and the main components thereof were 120 to 130 kDa for the fragment expressed protein and 110 to 120 kDa for the fragment expressed protein. In addition, although an antibody reaction was observed in addition to the above band, it is considered that the antibody was a fragment of the expressed protein or an inclusion body of the expressed protein.

(3) Antitumor activity test An antitumor activity test was carried out on the soluble fraction of the disrupted bacterial cells (in the presence and absence of IPTG) obtained in (2) according to the method described in Example 2. . The results were as shown in FIG. Both the DNA fragment and the expressed protein showed a selective antitumor activity.

[00101]

[Sequence List] SEQUENCE LISTING <110> New Food Creation <120> Anti-tumor protein from Pleurotus cornucopiae and gene encoding same <130> 11996601 <140> <141> <150> JP / 215311/1997 <151> 1997-08 -08 <150> JP / 66176/1998 <151> 1998-03-02 <160> 3 <170> PatentIn Ver. 2.0 <210> 1 <211> 633 <212> PRT <213> Pleurotus cornucopiae <400> 1 Met Ser Ile Gly Ser Asn Asp Pro Phe Tyr Asn Phe Ser Lys Ser Asp 1 5 10 15 Trp Lys Ser Lys Ser Ala Ala Thr Ile Ala Pro Pro Ala Gly Gln Pro 20 25 30 Tyr Arg Gly Leu Pro Leu Pro Pro Leu Pro Trp Ile Pro Gly Lys Leu 35 40 45 Pro Ser Gly Asp Thr Ser Lys Phe Asp Val Val Ile Val Gly Ser Gly 50 55 60 Pro Val Gly Ser Thr Tyr Ala Arg Leu Leu Val Glu Ala Gly Phe Lys 65 70 75 80 Val Gly Met Phe Glu Ile Gly Glu Ile Asp Ser Gly Arg Lys Leu Gly 85 90 95 Ser His Lys Lys Asn Thr Val Glu Tyr Gln Lys Asn Ile Asp Lys Phe 100 105 110 Val His Val Ile Gln Gly Gln Leu Met Pro Val Ser Val Pro Val Asn 115 120 125 Lys Tyr Val Ala Asp Thr Leu Ser Pro Ala Ser Trp Gln Ala Ser Thr 130 135 140 His Phe Val Arg Asn Gly Ala Asn Pro Glu Gln Asp Pro Phe Thr Asn 145 150 155 160 Leu Ser Gly Gln Ala Val Thr Arg Val Val Gly Gly Met Ala Thr His 165 170 175 Trp Thr Cys Ala Thr Pro Arg Phe His Lys Ser Glu Arg Pro Lys Leu 180 185 190 Val Lys Asp Asp Asp Ser Ala Asp Ala Ile Glu Trp Glu Arg Leu Tyr 195 200 205 Asp Ile Ala Glu Ser Phe Val Lys Thr Gly His Asn Gln Phe Asp Gln 210 215 220 Ser Ile Arg His Asn Leu Val Leu Glu Lys Leu Gln Glu Ser Tyr Ala 225 230 235 240 Gly Gln Arg Gly Phe Glu Gln Ile Pro Leu Ala Ala Gln Arg Thr Asn 245 250 255 Pro Arg Phe Val Glu Trp Ser Ser Ala His Thr Val Phe Asp Leu Glu 260 265 270 Asn Arg Pro Asn Ala Asp Asp Glu Lys Gly Arg Phe Asn Leu Phe Pro 275 280 285 Ala Val Val Cys Glu Arg Val Thr Arg Asp Ser Leu Asp Arg Lys Ile 290 295 300 Glu Asp Ile Glu Val His Asp Leu Ile Ser Gly Asp Arg Tyr Lys Val 305 310 315 320 Lys Ala Asp Val Phe Ile Leu Cys Ser Gly Ala Val His Asn Pro Gln 325 330 335 Ile Leu Val Asn Ser Gly Phe Gly Arg Met Gly Gln Pro Asp Ser Ser 340 345 350 Leu Pro Pro Pro Thr Leu Leu Pro Tyr Leu Gly Ser Tyr Ile Thr Glu 355 360 365 Gln Thr Leu Thr Phe Cys Gln Thr Val Phe Ser Thr Glu Leu Val Asn 370 375 380 Leu Val Lys Ser Asp Met Ile Ile Val Gly Thr Pro Gly Gln Pro Asp 385 390 395 400 400 Tyr Ser Val Thr Phe Thr Pro Asp Ser Pro Ser Asn Lys His Pro Asn 405 410 415 Trp Trp Asn Glu Lys Val Gln Lys His Met Met Gln His Gln Glu Asp 420 425 430 Pro Leu Pro Ile Pro Leu Asp Asp Pro Glu Pro Gln Val Thr Thr Leu 435 440 445 Phe Gln Asp Thr His Pro Trp His Thr Gln Ile His Arg Asp Ala Phe 450 455 460 Ser Tyr Gly Ala Val Ala Glu Ser Ile Asp Ser Arg Leu Val Val Asp 465 470 475 480 Trp Arg Phe Phe Gly Arg Thr Glu Pro Val Glu Glu Asn Lys Leu Trp 485 490 495 Phe Ser Lys Gln Ile Thr Asp Ala Tyr Asn Leu Pro Gln Pro Thr Phe 500 505 510 Ser Phe Arg Phe Pro Gln Gly Arg Thr Ala Gln Glu Ala Glu Leu Met 515 520 525 Met Ala Asp Met Cys Thr Met Ser Thr Lys Val Gly Gly Phe Leu Pro 530 535 540 Gly Ser Tyr Pro Gln Phe Met Ala Pro Gly Leu Val Leu His Leu Gly 545 550 555 56 0 Gly Thr His Arg Met Gly Phe Asp Glu Glu Ala Asp Lys Ala Cys Val 565 570 570 Asp Thr Asn Ser Lys Val Phe Gly Met Glu Asn Leu Phe Leu Gly Gly 580 585 590 Cys Gly Asn Ile Gly Thr Ala Tyr Ala Ser Asn Pro Thr Leu Thr Ala 595 600 605 Val Ala Leu Ala Ile Arg Ser Cys Lys Tyr Ile Arg Asn Asn Phe Thr 610 615 620 Pro Ser Pro Val Thr Gly Pro Asn Ala 625 630 <210> 2 <211> 1902 <212> DNA <213> Pleurotus cornucopiae <400> 2 atgtctattg gctcaaacga ccccttctac aacttttcaa agagcgattg gaagagcaag 60 tcagcggcga ccattgctcc gcccgccggt cagccatacc gtggcttgcc acttccaccg 120 ttgccttgga ttccgggaaa acttccgtcg ggagatacat ccaagttcga cgttgtcatc 180 gtcggctctg gcccagttgg ctctacttat gcccgtctcc tggtcgaggc aggtttcaaa 240 gtaggtatgt ttgaaattgg agaaatcgac tctggccgca agcttggttc ccataagaaa 300 aatacggttg agtaccagaa gaacattgac aaattcgtcc atgtcattca aggacaactc 360 atgcctgtat ccgtccctgt caacaaatat gttgccgata ctctcagccc cgctagctgg 420 caggcctcaa cacactttgt ccgcaacggc gcaaacccgg agcaagatcc ctttactaat 480 ctctcaggcc aagctgtgac ccgc gttgtg ggaggaatgg cgacgcactg gacttgcgca 540 acacctcgct tccacaagag tgagcggcct aagcttgtga aagatgatga ctccgcggac 600 gccatcgagt gggaacgcct ctatgacatc gccgaatcgt tcgtcaagac cgggcataat 660 cagtttgatc aatctatccg gcacaacctt gtactcgaga agcttcagga gtcctatgca 720 ggtcagcgcg gatttgagca gatccctctt gcggcccaac gcaccaaccc ccgcttcgtc 780 gagtggagct ctgcgcatac ggtctttgac cttgagaacc gcccaaacgc ggatgacgag 840 aaggggcgtt tcaacctctt ccccgcagtt gtatgtgaac gcgttacgcg ggattccctc 900 gaccggaaga tagaggacat tgaggtccat gacctgatta gcggcgatcg ctacaaggtc 960 aaggctgacg tgtttatcct ttgttctggc gcagtccata atccccagat tcttgtaaac 1020 tctggattcg ggcgcatggg tcagccagat tcttcactgc caccgccgac tctgctgccg 1080 tatctcggta gctacatcac tgagcagaca ctcacgttct gccagaccgt cttcagcacc 1140 gaactcgtca atcttgtcaa gtcggacatg attattgttg gcacgcccgg acagccagac 1200 tacagtgtta ctttcacccc ggacagtccg agcaataagc acccaaattg gtggaacgag 1260 aaggtccaga agcacatgat gcagcaccag gaggatcccc tcccgatccc gctcgatgac 1320 cctgagccac aggtcacgac actcttccag gacacac atc catggcacac tcagattcat 1380 cgtgatgcct ttagctacgg cgctgtggcg gagagtatcg acagccgtct tgttgttgac 1440 tggcgcttct tcggtcgcac tgaacctgtg gaggagaaca agctgtggtt ctcaaaacag 1500 atcactgatg catacaacct gccacagccc acattcagct tccggttccc ccagggccgc 1560 acagcccagg aggccgagct catgatggct gacatgtgca cgatgtcaac caaagtcggt 1620 ggcttcctgc ccggttcata tccgcagttt atggctcctg gccttgtact ccaccttggt 1680 ggaacccacc gcatgggctt tgatgaggaa gctgataagg cgtgtgtcga caccaactca 1740 aaggtcttcg gcatggagaa cctgttcctt ggcggttgcg gcaacattgg caccgcgtat 1800 gcctcgaacc cgacgctcac tgcggttgcg cttgcaatca ggagctgtaa gtatatcagg 1860 aataatttca cgccaagtcc tgtcactggg cccaatgcct aa 1902 <210> 3 <211> 50 <212> Lesa Pro Puls Phe Val Gly 1 5 10 15 Asp Gly Pro Val Gly Ser Thr Tyr Ala Arg Leu Cys Val Glu Ala Gly 20 25 30 Phe Lys Val Gly Met Phe Glu Lys Gly Glu Met Asp Ser Gly Arg Lys 35 40 45 Leu Gly 50

[Brief description of the drawings]

FIG. 1 is a diagram showing a method for preparing a test substance for each mushroom.

FIG. 2 is a diagram showing a method for preparing an antitumor protein derived from Pleurotus cornucopia.

FIG. 3 is a diagram showing a procedure for measuring antitumor activity.

FIG. 4 is a view showing a pattern of elution of a 40% ammonium sulfate precipitate using a TSKgel DEAE Toyopearl 650M column. The antitumor activity of each fraction is shown in the upper graph.

FIG. 5: TSKgel Phenyl Toyopearl 650
FIG. 3 is a view showing an elution pattern of an active fraction of anion exchange chromatography using an M column. The antitumor activity of each fraction is shown in the upper graph.

FIG. 6 is a view showing an elution pattern of a hydrophobic chromatography-active fraction on a Sepharose CL-6B column. The antitumor activity of each fraction is shown in the upper graph.

FIG. 7 is an electrophoresis photograph showing an SDS-polyacrylamide electrophoresis pattern (7.5% gel) of a gel filtration active fraction. The temperature display indicates the processing temperature of the sample.

FIG. 8 shows that the gel filtration active fraction was subjected to TSKgel G3000.
FIG. 4 is a diagram showing the results of measuring the molecular weight by a SW _XL column.

FIG. 9 is a view showing an ultraviolet absorption spectrum of a gel filtration active fraction.

FIG. 10 shows a gel filtration active fraction as a sample at 4 ° C. to 10 ° C.
(A) and the electrophoresis photograph (B) which showed the result of having tested the stability with respect to heat up to 0 degreeC.

FIG. 11 shows a gel filtration active fraction as a sample, from 1 to 12.
(A) and (B) are electrophoretic photographs showing the results of testing the stability to pH up to 5.

FIG. 12 shows the results of analyzing the N-terminal amino acid sequence of a protein (62 kDa) transferred to a PVDF membrane after subjecting a gel filtration active fraction to SDS-polyacrylamide electrophoresis.

FIG. 13 shows the concentration of gel filtration active fraction and cells (BALB / 3T3 c
FIG. 2 is a view showing the relationship between lone A31 and SV-T2) growth inhibition.

FIG. 14 is a view showing primers used for synthesizing a gene according to the present invention.

FIG. 15 is an electrophoretic photograph showing the results of Western blot analysis of a soluble fraction and an insoluble fraction obtained by disrupting Escherichia coli (Example 5) transformed with a vector containing a DNA fragment. (A) shows the case where a DNA fragment was used, and (B) shows the case where a DNA fragment was used. Lanes 3 to 6 show heat-treated samples, and lanes 7 to 10 show samples without heat treatment. Also,
Lanes 3, 5, 7, and 9 are the soluble fraction, lane 4,
6, 8, and 10 indicate insoluble fractions. Lane 3,
4, 7, and 8 show proteins expressed under IPTG induction, and lanes 5, 6, 9, and 10 show proteins expressed without IPTG induction. Lanes 1 and 2 show the antitumor protein purified from Euglena (Example 1). Lane 1 is a sample subjected to heat treatment, and lane 2 is a sample not subjected to heat treatment. Arrows indicate antitumor proteins. The lower arrow indicates a monomer, and the upper arrow indicates a dimer (presumed to be a dimer or tetramer).

FIG. 16 shows the antitumor activity of a solubilized fraction obtained by disrupting transformed E. coli. (A) is DN
(B) shows the case where the A fragment was used and the case where the DNA fragment was used. ●: BALB / 3T3 clone A31 (with IPTG), ○: BALB / 3T3
Clone A31 (without IPTG), ★: SV-T2 (I
PTG), ☆: SV-T2 (no IPTG).

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Kazuaki Kido 410 Akanuma-do, Kasukabe-shi, Saitama Prefecture Inside the Momoya Research Laboratory Co., Ltd. (72) Inventor Tomohide Saka 410 Akanuma-do, Kasukabe-shi, Saitama Momoya, Inc. Inside the laboratory (72) Inventor, Ryoko Aoki 410, Akanuma-do, Kasukabe, Saitama Pref.

Claims (19)

[Claims] 1. An antitumor protein derived from the fruiting body of Pleurotus cornucopia having the following properties (1) to (5): (1) salted out by 60% ammonium sulfate, and (2) an estimated molecular weight by gel filtration. 150-200 kDa, molecular weight estimated by HPLC is about 200 kDa, (3) maximum absorption is observed at a wavelength of around 277 nm in the ultraviolet absorption spectrum, (4) precipitation at a temperature exceeding 60 ° C., and antitumor activity is reduced. (5) It has a stable antitumor activity in the pH range of 3 to 12. 2. The antitumor protein according to claim 1, which has an amino acid composition ratio shown in the following table. [Table 1] 3. An amino acid sequence comprising the amino acid sequence of SEQ ID NO: 1.
protein. 4. A protein comprising one or more additions, insertions, substitutions, and / or deletions and having the amino acid sequence of SEQ ID NO: 1 having antitumor activity. 5. The protein according to claim 4, wherein the amino acid sequence of SEQ ID NO: 1 has a deletion of one or more amino acid residues selected from amino acid residues 1 to 46. 6. The amino acid sequence of SEQ ID NO.
5. The protein of claim 4, which has a deletion of consecutive amino acid residues of from 1 to 46). 7. The protein according to claim 4, wherein the amino acid sequence of SEQ ID NO: 1 has a deletion of amino acid residues 1 to 46. 8. A nucleotide sequence encoding the protein according to any one of claims 3 to 7. 9. A composition comprising the nucleotide sequence of SEQ ID NO: 2.
A nucleotide sequence according to claim 8. 10. A peptide comprising the amino acid sequence of positions 47 to 96 of SEQ ID NO: 1. 11. A vector comprising the nucleotide sequence according to claim 8 or 9. 12. The vector according to claim 11, which is selected from the group consisting of a plasmid vector, a virus vector, and a liposome vector. (13) A host cell transformed with the nucleotide sequence of (8) or (9) or the vector of (11) or (12). 14. The host cell according to claim 13, which is selected from the group consisting of Escherichia coli, yeast, Bacillus subtilis, insect cells, animal cells, and plant cells. 15. The method according to claim 3, comprising culturing the host cell according to claim 13 or 14, and isolating the protein according to any one of claims 3 to 7 from the culture. 8. The method for producing the protein according to any one of items 7 to 7. 16. A pharmaceutical composition comprising the protein according to claim 1 together with a pharmaceutically acceptable carrier. 17. The method according to claim 1, which is used for treating a malignant tumor.
7. The pharmaceutical composition according to 6. (18) an antibody against the protein of (1); 19. The method according to claim 18, which is a polyclonal antibody.
The antibody according to 1.